The broad long-term objectives of this proposal are to identify the molecular determinants of psychostimulant recognition (cocaine and amphetamine) by serotonin transporters (SERTs), thereby revealing fundamental mechanisms involved SERT function and modulation by drugs. This project has direct health relatedness as SERTs are the molecular target for.most antidepressant drugs as well as many drugs of abuse. Antidepressants such as paroxetine, fluoxetine, and imipramine bind to the transporter and inhibit serotonin uptake, thereby prolonging the extracellular lifespan of the neurotransmitter. The abused psychostimulants cocaine and amphetamine also bind to the transporter, but have distinct pharmacologic effects leading to abuse potential and possible neurotoxicity. Despite the cloning of SERT in the early 1990's, the molecular and cellular neurobiology surrounding the function of this transport protein remains to be fully elucidated. This project uses a multidisciplinary approach aimed at significantly advancing knowledge regarding the molecular pharmacology of psychostimulants at SERTs. The studies will use multiple techniques including expression and characterization of recombinant transporters in mammalian cells, the substituted cysteine accessibility method (SCAM), real-time fluorescent substrate assays, electrophysiology, and site-directed mutagenesis to identify amino acids involved with specific transporter functions including antagonist and substrate binding. In addition, computer-assisted structure-activity studies of cocaine and amphetamine derivatives will be coupled with mutagenesis in an attempt to identify direct ligand-transporter interactions. Therefore, the specific aims of this project are: 1) To determine the core domains forming the substrate permeation pathway, 2) To elucidate information about the orientation of SERT domains, and 3) To identify and elucidate the role of amino acids in the core domains involved with recognition of psychostimulants. The proposed strategies will provide critical new information linking protein structure to functional properties of the transporter and an enhanced understanding of the molecular mechanisms of action for psychotherapeutic and abused drugs. Abused stimulants such as cocaine and amphetamine (including methamphetamine ("Meth") and MDMA or 'ecstasy') are of great societal concern. The proposed studies will reveal important new information on how these abused drugs alter their target in the brain (i.e., the serotonin transporter) and offer new strategies to combat the addictive properties of these substances.